京都大学 / 新制・論文博士 / 博士(医学) / 乙第13609号 / 論医博第2319号 / 新制||医||1073(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村川 泰裕, 教授 竹内 理, 教授 永井 純正 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Angiogênese é o processo de formação de novos vasos sanguíneos a partir dos vasos existentes. É um processo vital, mas muitas doenças também dependem deste mecanismo para obter nutrientes e progredir. Estas \"doenças dependentes de angiogênese\" incluem cânceres, retinopatias e degeneração macular. Alguns inibidores da angiogênese foram desenvolvidos na última década, com o objetivo de auxiliar no manejo dessas doenças e melhorar a qualidade de vida dos pacientes. A maioria destes compostos funciona inibindo a ligação de VEGFA/VEGFR2, que também é um elemento importante para a sobrevivência de células endoteliais quiescentes; e isso pode explicar parcialmente eventos adversos observados em alguns ensaios clínicos. Nossa hipótese é que a melhoria das terapias anti-angiogênicas depende de uma compreensão melhor e mais ampla desse processo, especialmente quando relacionada à progressão das doenças. Utilizando RNA-Seq e um modelo animal bem aceito de angiogênese, o modelo murino de Retinopatia Induzida por Oxigênio, exploramos o transcritoma e identificamos 153 genes diferencialmente expressos durante a angiogênese. Uma extensiva validação de vários genes realizada por qRT-PCR e hibridização in-situ confirmou a superexpressão de Esm1 em células endoteliais de tecidos com angiogênese ativa. A análise de enriquecimento desta lista de genes confirmou a ligação da angiogênese com genes frequentemente mutados em tumores, consistente com a conhecida ligação entre câncer e angiogênese, e forneceu sugestões de fármacos já aprovados que podem ser reutilizados para controlar a angiogênese em circunstâncias patológicas. Finalmente, com base neste panorama amplo da angiogênese, fomos capazes de criar um biomarcador molecular com poder prognóstico para a predição da recidiva de câncer de mama, com aplicações clínicas promissoras. Em resumo, este trabalho revelou com sucesso genes relacionados à angiogênese e forneceu novas alternativas terapêuticas, incluindo potenciais fármacos para reposicionamento. Esse conjunto de genes diferencialmente expressos é também um recurso valioso para investigações futuras. / Angiogenesis is the process of formation of new blood vessels based on existing vessels. It is a vital process but many diseases also rely on this mechanism to get nourishment and progress. These so called angiogenesis-dependent diseases include cancers, retinopathies and macular degeneration. Some angiogenesis inhibitors were developed in the past decade, aiming to help the management of such diseases and improve patients quality of life. Most of these compounds work by inhibiting VEGFA/VEGFR2 binding, which is also a key element to the survival of quiescent endothelial cells; this may partly explain unanticipated adverse events observed in some clinical trials. We hypothesize that the improvement of anti-angiogenesis therapies hinges on a better and broader understanding of the process, especially when related to diseases\' progression. Using RNA-seq and a well accepted animal model of angiogenesis, the murine model of Oxygen Induced Retinopathy, we have explored the transcriptome landscape and identified 153 genes differentially expressed in angiogenesis. An extensive validation of several genes carried out by qRT-PCR and in-situ hybridization confirmed Esm1 overexpression in endothelial cells of tissues with active angiogenesis, providing confidence on the results obtained. Enrichment analysis of this gene list endorsed a narrow link of angiogenesis and frequently mutated genes in tumours, consistent with the known connection between cancer and angiogenesis, and provided suggestions of already approved drugs that may be repurposed to control angiogenesis under pathological circumstances. Finally, based on this comprehensive landscape of angiogenesis, we were able to create a prognostic molecular biomarker for prediction of breast cancer relapse, with promising clinical applications. In summary, this work successfully unveiled angiogenesis-related genes, providing novel therapeutic alternatives, including potential drugs for repositioning. The set of differentially expressed genes is also a valuable resource for further investigations.

Deux phénomènes liés au TFIIH ont été étudiés : l'influence des mutations spécifiques dans la sous-unité XPD de TFIIH sur la réponse transcriptionnelle de certains gènes après l'irradiation UV, et l'interaction entre le TFIIH et la transcription des gènes de classe III. Une analyse détaillée de la dynamique du transcriptome a été effectuée pour la réponse des cellules humaines mutantes XP-D/CS à l'UV. Il a été démontré que la dysrégulation sélective observée de l’expression des gènes était liée à l'incapacité pour la ré-initiation transcriptionnelle et à l'hétérochromatinisation suivante, où l'histonedésacétylase SIRT1 a été identifiée comme le principal facteur. Son inhibition a permis de recouvrer l'expression normale d'un nombre substantiel des gènes affectés. Une étude de la participation pangénomique du coeur de TFIIH dans latranscription a découvert son association avec les gènes actifs de classe III. Cette association a été démontrée être indépendante de Pol II. Le coeur de TFIIH a été montré participer directement à la transcription effectuée in vitro par Pol III. / In this work, two TFIIH-related phenomena were investigated : the influence of specific mutations in TFIIH XPD subunits on the transcriptional response of different genes on UV irradiation and the interaction between TFIIH and transcription of class III genes. For the first time the detailed investigation of transcriptome dynamics was carried out for the response of XP-D/CS mutant human cells to UV-irradiation. The transcription regulation nature of the observed selective gene expression dysregulation was clearly observed. Its relation to failure of transcription re-initiation and consequentheterochromatisation was demonstrated. SIRT1 histone deacetylase was identified as the main driver of the repressive chromatin establishment on the certain genes upon UV. Inhibition of SIRT1 was found to recover normal expression of substantial number of affected genes. SIRT1 mediated mechanism was shown to be XP-D/CS specific. A potential link between this longevity related protein and progeria features of XP-D/CS mutants was hypothesised. Genome-wide study of the involvement of the core TFIIH in transcription revealed its association with active class III genes, not described previously. This association was demonstrated to be Pol II-independent. The core TFIIH was shown to be directly involved in Pol III mediated transcription in vitro.

Le nombre de gènes codant pour des protéines chez l’'homme, le vers rond et la mouche des fruits est du même ordre de grandeur. Cette absence de correspondance entre le nombre de gènes d’un eucaryote et sa complexité phénotypique s’explique en partie par le caractère alternatif de l’épissage.L'épissage alternatif augmente considérablement le répertoire fonctionnel de protéines codées par un nombre limité de gènes. Ce mécanisme, très actif lors du développement embryonnaire, participe au devenir cellulaire. De nombreux troubles génétiques, hérités ou acquis (en particulier certains cancers), se caractérisent par une altération de son fonctionnement.Les technologies de séquençage à haut débit de l'ARN donnent accès a une information plus riche sur le mécanisme de l’épissage. Cependant, si la lecture à haut débit des séquences d’ARN est plus rapide et moins coûteuse, les données qui en sont issues sont complexes et nécessitent le développement d’outils algorithmiques pour leur interprétation. En particulier, la reconstruction des transcrits alternatifs requiert une étape de déconvolution non triviale.Dans ce contexte, cette thèse participe à l'étude des événements d'épissage et des transcrits alternatifs sur des données de séquençage à haut débit de l'ARN.Nous proposons de nouvelles méthodes pour reconstruire et quantifier les transcrits alternatifs de façon plus efficace et précise. Nos contributions méthodologiques impliquent des techniques de régression parcimonieuse, basées sur l'optimisation convexe et sur des algorithmes de flots. Nous étudions également une procédure pour détecter des anomalies d'épissage dans un contexte de diagnostic clinique. Nous suggérons un protocole expérimental facilement opérant et développons de nouveaux modèles statistiques et algorithmes pour quantifier des événements d’épissage et mesurer leur degré d'anormalité chez le patient. / The number of protein-coding genes in a human, a nematodeand a fruit fly are roughly equal.The paradoxical miscorrelation between the number of genesin an organism's genome and its phenotypic complexityfinds an explanation in the alternative natureof splicing in higher organisms.Alternative splicing largely increases the functionaldiversity of proteins encoded by a limitednumber of genes.It is known to be involved incell fate decisionand embryonic development,but also appears to be dysregulatedin inherited and acquired human genetic disorders,in particular in cancers.High-throughput RNA sequencing technologiesallow us to measure and question splicingat an unprecedented resolution.However, while the cost of sequencing RNA decreasesand throughput increases,many computational challenges arise from the discrete and local nature of the data.In particular, the task of inferring alternative transcripts requires a non-trivial deconvolution procedure.In this thesis, we contribute to deciphering alternative transcript expressions andalternative splicing events fromhigh-throughput RNA sequencing data.We propose new methods to accurately and efficientlydetect and quantify alternative transcripts.Our methodological contributionslargely rely on sparse regression techniquesand takes advantage ofnetwork flow optimization techniques.Besides, we investigate means to query splicing abnormalitiesfor clinical diagnosis purposes.We suggest an experimental protocolthat can be easily implemented in routine clinical practice,and present new statistical models and algorithmsto quantify splicing events and measure how abnormal these eventsmight be in patient data compared to wild-type situations.

Angiogênese é o processo de formação de novos vasos sanguíneos a partir dos vasos existentes. É um processo vital, mas muitas doenças também dependem deste mecanismo para obter nutrientes e progredir. Estas \"doenças dependentes de angiogênese\" incluem cânceres, retinopatias e degeneração macular. Alguns inibidores da angiogênese foram desenvolvidos na última década, com o objetivo de auxiliar no manejo dessas doenças e melhorar a qualidade de vida dos pacientes. A maioria destes compostos funciona inibindo a ligação de VEGFA/VEGFR2, que também é um elemento importante para a sobrevivência de células endoteliais quiescentes; e isso pode explicar parcialmente eventos adversos observados em alguns ensaios clínicos. Nossa hipótese é que a melhoria das terapias anti-angiogênicas depende de uma compreensão melhor e mais ampla desse processo, especialmente quando relacionada à progressão das doenças. Utilizando RNA-Seq e um modelo animal bem aceito de angiogênese, o modelo murino de Retinopatia Induzida por Oxigênio, exploramos o transcritoma e identificamos 153 genes diferencialmente expressos durante a angiogênese. Uma extensiva validação de vários genes realizada por qRT-PCR e hibridização in-situ confirmou a superexpressão de Esm1 em células endoteliais de tecidos com angiogênese ativa. A análise de enriquecimento desta lista de genes confirmou a ligação da angiogênese com genes frequentemente mutados em tumores, consistente com a conhecida ligação entre câncer e angiogênese, e forneceu sugestões de fármacos já aprovados que podem ser reutilizados para controlar a angiogênese em circunstâncias patológicas. Finalmente, com base neste panorama amplo da angiogênese, fomos capazes de criar um biomarcador molecular com poder prognóstico para a predição da recidiva de câncer de mama, com aplicações clínicas promissoras. Em resumo, este trabalho revelou com sucesso genes relacionados à angiogênese e forneceu novas alternativas terapêuticas, incluindo potenciais fármacos para reposicionamento. Esse conjunto de genes diferencialmente expressos é também um recurso valioso para investigações futuras. / Angiogenesis is the process of formation of new blood vessels based on existing vessels. It is a vital process but many diseases also rely on this mechanism to get nourishment and progress. These so called angiogenesis-dependent diseases include cancers, retinopathies and macular degeneration. Some angiogenesis inhibitors were developed in the past decade, aiming to help the management of such diseases and improve patients quality of life. Most of these compounds work by inhibiting VEGFA/VEGFR2 binding, which is also a key element to the survival of quiescent endothelial cells; this may partly explain unanticipated adverse events observed in some clinical trials. We hypothesize that the improvement of anti-angiogenesis therapies hinges on a better and broader understanding of the process, especially when related to diseases\' progression. Using RNA-seq and a well accepted animal model of angiogenesis, the murine model of Oxygen Induced Retinopathy, we have explored the transcriptome landscape and identified 153 genes differentially expressed in angiogenesis. An extensive validation of several genes carried out by qRT-PCR and in-situ hybridization confirmed Esm1 overexpression in endothelial cells of tissues with active angiogenesis, providing confidence on the results obtained. Enrichment analysis of this gene list endorsed a narrow link of angiogenesis and frequently mutated genes in tumours, consistent with the known connection between cancer and angiogenesis, and provided suggestions of already approved drugs that may be repurposed to control angiogenesis under pathological circumstances. Finally, based on this comprehensive landscape of angiogenesis, we were able to create a prognostic molecular biomarker for prediction of breast cancer relapse, with promising clinical applications. In summary, this work successfully unveiled angiogenesis-related genes, providing novel therapeutic alternatives, including potential drugs for repositioning. The set of differentially expressed genes is also a valuable resource for further investigations.

La viticulture dépend des conditions climatiques. Dans le contexte de réchauffement climatique, les changements de la vigne et du raisin sous l’effet des températures élevées vraisemblables pour les prochaines décennies pourraient modifier l’aire de répartition des cépages et même menacer la durabilité de la viticulture des régions chaudes. L’objectif de cette étude était donc d’analyser les effets de l’élévation de température sur la composition du raisin, du transcriptome au métabolome. L’utilisation d’un système « open top » passif au vignoble a permis d’augmenter la température autour des grappes de 0.5-1.6 °C en moyenne, une valeur compatible avec le réchauffement climatique prévisible. Les expérimentations ont été conduites sur Cabernet Sauvignon (CS) et Sauvignon Blanc (SB), de la nouaison à la surmaturité, à Bordeaux (France) et en Barossa Valley (Australie). Les analyses ont ciblé essentiellement les métabolites primaires, les composés phénoliques et aromatiques (l’IBMP, arôme de poivron vert; les précurseurs de 3SH, arôme de pamplemousse et la ß-damascenone, arômes floraux). En complément, des analyses RNA-seq et q-PCR ont été réalisées pour explorer la réponse transcriptomique à cet échauffement modéré en conditions réalistes de vignoble.L’échauffement modéré a peu affecté les concentrations en sucres, acides et total en acides aminés, mais a modifié la distribution des différents acides aminés. La composition en acides aminés s’est principalement différenciée suivant la variété, le stade de développement et le site expérimental.Les concentrations finales en IBMP n’ont pas été affectées par l’échauffement. Cependant, à la fermeture de la grappe, les baies de CS échauffées avaient une moindre concentration en IBMP associée à une sous-expression de VviOMT3. La concentration en IBMP des baies de SB échauffées n’a pas montré de différenciation, bien que les niveaux d’expression de VviOMT3 et VviOMT4 soient diminués. Les effets limités et dépendant du génotype suggèrent qu’une augmentation modérée de la température ne serait pas suffisante pour modifier significativement l’IBMP.Glut-3SH-Al était bien plus concentrée que Glut-3SH et Cys-3SH. le traitement échauffé a fait diminuer la présence de Glut-3SH-Al et Cys-3SH dans les baies de SB, en association avec une sous-expression de VviGST4. Par ailleurs, VIT_08s0007g01420 (GSTU8) a été réprimé par le traitement, et pourrait donc être un gène candidat potentiel impliqué dans la biosynthèse de précurseurs de 3SH.Pour les baies de CS, les concentrations en caroténoïdes totaux et celles des deux caroténoïdes majeurs (la lutéine et le β-carotène) n’ont pas réagi à l’augmentation de température. La zéaxanthine a montré une tendance à la diminution sous l’effet de l’échauffement, jusqu’à une diminution significative. Cette concentration plus faible pourrait limiter la biosynthèse de β-damascenone et expliquer la plus faible teneur en β-damascenone observée dans les baies à sur-maturité en cas de température élevée.Un total de 357 gènes (DEGs) ont été différentiellement exprimés en réponse à l’augmentation de température pour les échantillons de 2015 à Bordeaux. D’après l’analyse d’enrichissement « Gene Ontology », le traitement a principalement régulé quatre catégories en relation avec les microtubules, la paroi cellulaire, l’espace extracellulaire et l’activité des facteurs de transcription. 6 DEGs liés à la biosynthèse des anthocyanes ont été régulés négativement, ce qui pourrait expliquer, au moins en partie, la concentration réduite en anthocyanes totaux observée dans les baies de CS échauffées. En revanche, les tanins n’ont pas été affectés par l’augmentation de la température. / Viticulture depends on climate conditions during the growing season. In the context of global warming, any changes in viticulture caused by the rising temperatures expected for the next decades may alter the geographical distribution of grape varieties and even threaten the sustainability of viticulture in hot areas. The objective of this research was to investigate the effects of moderately elevated temperature on grape composition, both at metabolic and transcriptomic levels. A passive open-top heating system was applied in Cabernet Sauvignon (CS) and Sauvignon Blanc (SB) vines grown with standard practice in Bordeaux, France and the Barossa Valley, Australia (CS only) to increase the bunch zone mean temperature by around 0.5-1.6 °C, which was commensurate with the projected global warming. This moderate heating was applied from fruit-set to two weeks after harvest. Metabolites related to technical, phenolic and aromatic maturities (IBMP, the green pepper aroma, precursors of 3SH, grapefruit aroma, and β-damascenone, floral aroma) were assessed, together with transcriptome analysis via RNA-seq and q-PCR, in order to obtain a comprehensive view of berry responses to this moderately elevated temperature in realistic vineyard conditions.The moderately elevated temperature hardly affected the concentrations of sugars, organic acids and total amino acids, but it altered free amino acid composition depending on varieties, vintages and locations.The final concentrations of IBMP were not affected by warming condition in mature berries. However, the elevated temperature significantly reduced IBMP content and expression level of VviOMT3 (a known key gene of IBMP) in CS berries at bunch closure stage, while it reduced the expression levels of VviOMT3 and VviOMT4 at bunch closure stage without affecting IBMP concentration in SB berries. This limited and genotype-dependent effect of elevated temperature suggests that a moderate temperature elevation may not be sufficient to significantly modify IBMP.Glut-3SH-Al was much more concentrated than Glut-3SH and Cys-3SH. Reduced Glut-3SH-Al and Cys-3SH concentrations were associated with a significantly lower expression level of VviGST4 in heated SB berries. Meanwhile, VIT_08s0007g01420 (GSTU8), was down-regulated by elevated temperature and might be a potential candidate gene involved in the biosynthesis of precursors of 3SH.The concentrations of total carotenoids and two predominant carotenoids (lutein and β-carotene) were not altered by elevated temperature in CS berries, but zeaxanthin was reduced by elevated temperature and was significantly less concentrated at harvest. This lower concentration may limit the biosynthesis of β-damascenone and explain the observed lower β-damascenone concentration in post-ripening berries under elevated temperature.A total of 357 genes were differentially expressed (DEGs) in response to the elevated temperature in Bordeaux samples in 2015. Enrichment analysis of Gene Ontology showed that temperature mainly regulated four GO categories, including microtubule, cell wall, extracellular region, and transcription factor activity. 6 DEGs related to anthocyanins synthesis were down-regulated and it could explain, at least in part, the observed lower total anthocyanins in warmed CS. Conversely, tannins were not affected by elevated temperature.The results provide a better understanding of potential global warming effects on metabolite changes during berry development, along with novel molecular insights into the response of grape berry to moderate heating in vineyard conditions.

Le glioblastome (GBM) est la tumeur primitive du cerveau la plus fréquente et la plus agressive chez l’adulte. Le mauvais pronostic de cette pathologie peut être expliqué par la présence de cellules résistantes aux traitements à l’origine des rechutes appelées cellules souches de glioblastome (CSG). Caractérisées par une plasticité cellulaire, elles sont capables de s’adapter aux environnements défavorables à leur survie. Ainsi, malgré des traitements multimodaux agressifs, les bénéfices de la prise en charge du GBM restent très modestes ; il est donc nécessaire de mieux caractériser ces cellules afin de développer de nouvelles thérapies ciblant les CSG. Le rôle des mécanismes post-transcriptionnels de régulation de l’expression génique dans le maintien des cellules souches cancéreuses a été démontré dans différentes tumeurs. Cependant, peu de protéines de liaison à l'ARN (RBP), régulateurs clés de ces événements, ont été identifiés dans le contexte des CSG. Mon projet de thèse s’inscrit dans le cadre de l’étude de RBP régulant les propriétés d’auto-renouvèlement et de survie des CSG afin d’approfondir nos connaissances sur les mécanismes moléculaires qui contribuent à la formation ou récurrence des GBM. Dans un premier temps, j’ai cherché à définir un protocole permettant d’enrichir la population de CSG maintenues en conditions de culture non-adhérentes qui favorisent la formation de neurosphères et le maintien d’une hiérarchie cellulaire. Confrontée à une forte hétérogénéité de signatures moléculaires, j’ai choisi de prendre cette caractéristique en compte dans un second temps en menant une étude comparative du transcriptome de 5 modèles de cellules souches de glioblastome provenant de patients différents. Cette analyse inclut également des cellules différenciées in vitro à partir des CSG ainsi que des cellules souches neurales humaines. Grâce à une approche de séquençage de leur transcriptome, mes travaux de thèse ont permis d’identifier une famille de régulateurs post-transcriptionnels enrichis dans les CSG, les protéines nELAVL. Ces résultats ont pu être confirmés par l’analyse de leur expression protéique dans des modèles in vitro et dans des coupes de tumeurs provenant de différents patients atteints de GBM. Une co-expression des protéines nELAVL avec OLIG2 ou SOX2 a été observée confirmant ainsi leur association avec l’état souche. ELAVL4 correspond au membre de la famille nELAVL le plus réprimé lors de la différenciation des CSG. Des outils de modulation de son expression ont été développé en vue d’évaluer son rôle dans les CSG par des approches de perte d’expression ou de gain de fonction / Glioblastoma (GBM) is the most common and aggressive primary adult brain tumor. GBM dismal prognosis can be explained by the presence of treatment resistant cells responsible for tumor relapse known as glioblastoma stem cells (GSC). Characterized by cellular plasticity, they are able to adapt to hostile environments. Thus, despite aggressive multimodal treatments, GBM curative therapies have provided only a modest benefit; it is therefore necessary to better characterize these cells in order to develop new GSC targeted therapies. The role of posttranscriptional mechanisms of gene expression regulation in the maintenance of cancer stem cells has been demonstrated in different tumors. However, few RNA-binding proteins (RBPs), key regulators of these events, have been identified in GSC. My thesis project consists in identifying RBP that are critical for self-renewal and increased survival properties of GSC. The aim of this study is to deepen our knowledge of the underlying molecular mechanisms of GBM development or recurrence. At first, I established a protocol to enrich for GSC using nonadherent culture conditions that promote the formation of neurospheres comprising a cellular hierarchy. I chose to take into account the facing problem of GSC molecular heterogeneity in a second step and carried out a comparative study of the transcriptome of 5 glioblastoma stem cell cultures from different patients. This analysis also includes in vitro differentiated cells originating from GSC as well as human neural stem cells. Using a transcriptome sequencing approach, my thesis work has led to the identification of a family of post-transcriptional regulators enriched in GSC, nELAVL proteins. These results were confirmed by protein expression analysis in in vitro neurospheres and within tumor sections from different GBM patients. Co-expression of nELAVL proteins with OLIG2 or SOX2 was observed thus confirming their association with stemness. ELAVL4 repesents the most differentially expressed member of nELAVL family upon GSC differentiation. Knock-down and gain-offunction tools targeting ELAVL4 have been developed to further assess its roles in GSC maintenance

L’acide rétinoïque (AR) agit via des récepteurs nucléaires (RAR) qui sont des facteurs de transcription inductibles par le ligand. Il active aussi des cascades de kinases qui ciblent les RAR et modulent leur activité transcriptionnelle. Cependant, l’ensemble des protéines phosphorylées en réponse à l’AR de même que les conséquences des dérégulations du « kinome » sur les effets l’AR et le fonctionnement des RAR demeurent mal connus. J’ai comparé les effets de l’AR sur le phosphoprotéome de deux lignées de cellules de cancer du sein : MCF7, qui est sensible à l’AR, et BT474, qui surexprime le récepteur a activité tyrosine kinase erbB-2 et est résistante à l’AR. De nombreuses différences ont été observées avec des répercussions sur l’expression des gènes de même que sur la phosphorylation, le recrutement aux promoteurs des gènes cibles et la dégradation de RAR alpha par le protéasome. J’ai aussi montré que la dégradation de RAR alpha met en jeu TRIM24 qui contrôle sa déubiquitination. / Retinoic acid (RA) acts by binding to specific nuclear receptors (RARs), which are ligand-dependant transcription factors. RA also has non-genomic effects and activates kinase cascades that target RARs and modulate their transcriptional activity. However, the proteins that are phosphorylated in response to RA remain to be identified. The consequences of dysregulations of the "kinome" on the non-genomic effects of RA and on RAR function also require further investigation. I compared the effect of RA on the phosphoproteome of two breast cancer cell lines: MCF7, which is RA-sensitive, and BT474, a RA-resistant cell line that overexpresses the receptor tyrosine kinase erbB-2. Multiple differences were observed with consequences on gene expression as well as on phosphorylation, recruitment on target genes promoters and RARalpha degradation by the proteasome. In the context or RARalpha degradation, I showed the involvement of TRIM24 which controls RARα deubiquitination.

Les Escherichia coli entérohémorragiques (EHEC) sont des E. coli producteurs de Shiga-toxines (STEC) représentant le quatrième agent responsable de toxi-infections alimentaires en Europe. La contamination par ces pathogènes résulte principalement de l’ingestion de produits alimentaires contaminés par les fèces de bovins, dont le tube digestif apparait comme le principal réservoir naturel des EHEC. Ces pathogènes survivent dans le tractus digestif du ruminant, qui est porteur sain, et semblent bien adaptés à l’ensemble de cet écosystème complexe. Réduire le portage animal est une stratégie de choix afin de limiter les toxi-infections humaines à EHEC. L’objectif de cette thèse était d’approfondir les connaissances sur la physiologie et l’écologie des EHEC dans le tube digestif du bovin, une étape primordiale pour proposer, à terme, différentes stratégies visant à limiter le portage. L’analyse du transcriptome de la souche EHEC O157:H7 de référence EDL933 a permis l’identification de voies métaboliques utilisées par les EHEC dans différents compartiments du tube digestif de l’animal. Certains sucres, dont ceux issus de la couche de mucus intestinal, et acides aminés ainsi que l’éthanolamine semblent représenter des substrats importants pour la survie des EHEC tout au long du tube digestif du bovin. Cette étude transcriptomique a également mis en évidence l’activation, par la souche EHEC, de nombreux systèmes de résistance à différents stress rencontrés dans le tube digestif bovin, dont les systèmes toxines/anti-toxines. L’activation de ces systèmes et la capacité à former des biofilms ont également été observées chez une souche STEC O157:H7 d’origine bovine, la souche MC2, dans des conditions mimant une persistance dans l’environnement. La caractérisation génomique et phénotypique permet de considérer cette souche comme pathogène et des études réalisées in vitro et in vivo ont indiqué que la souche MC2 était capable de persister dans le tube digestif du bovin mais aussi dans l’environnement de l’élevage. L’inoculation expérimentale de bovins par la souche MC2 a permis de mettre au point le premier modèle animal reproductible de portage et d’excrétion des STEC O157:H7 décrit en France. Ce modèle pourra être utilisé pour tester in vivo l’effet d’additifs alimentaires, tels que les probiotiques, afin de réduire le portage et l’excrétion de souches EHEC par les bovins, et donc limiter la contamination de l’Homme. / Enterohaemorrhagic Escherichia coli (EHEC) are Shiga-toxin producing E. coli (STEC) which represent the fourth pathogen leading to foodborne illness in Europe. Contamination by these pathogens results mainly from the ingestion of food contaminated by feces of bovine, for which the digestive tract appears as the main natural reservoir of EHEC. These pathogens survive in the digestive tract of ruminants, which is healthy carriers, and seem well-adapted to this complex ecosystem. Reducing animal carriage is a strategy of choice to limit EHEC human infections. The aim of this thesis was to increase our knowledge on the physiology and ecology of EHEC in the digestive tract of bovine, a key step to propose, ultimately, different strategies to limit the carriage. Transcriptome analysis of the EHEC O157:H7 reference strain EDL933 allowed the identification of metabolic pathways used by EHEC in different compartments of the digestive tract of the animal. Some carbohydrates, including those from the intestinal mucus layer, and amino acids as well as ethanolamine appear to be important substrates for the survival of EHEC throughout the bovine digestive tract. This transcriptomic study also revealed the activation, by the EHEC strain, of several stress resistance systems encountered in the bovine digestive tract, including toxin/anti-toxin systems. The activation of these systems and the ability to form biofilms have also been observed in a bovine STEC O157:H7 strain, MC2 strain, under conditions mimicking persistence in the environment. Genomic and phenotypic characterization allows this strain to be considered as pathogenic and in vitro and in vivo studies indicated that the MC2 strain was able to persist in the bovine digestive tract but also in the farm environment. The experimental inoculation of bovines with the MC2 strain led to the development, for the first time in France, of a reproducible animal model of carriage and excretion of STEC O157:H7. This model could be used to test in vivo the effect of food additives, such as probiotics, in order to reduce the carriage and excretion of EHEC strains by bovines, and thus limit the contamination of humans.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-25T15:11:49Z No. of bitstreams: 2 Dissertação - Mariane Brom Sobreiro - 2017.pdf: 16013047 bytes, checksum: 6d41859718e06adc6294f5de038bc631 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-25T15:12:31Z (GMT) No. of bitstreams: 2 Dissertação - Mariane Brom Sobreiro - 2017.pdf: 16013047 bytes, checksum: 6d41859718e06adc6294f5de038bc631 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-25T15:12:31Z (GMT). No. of bitstreams: 2 Dissertação - Mariane Brom Sobreiro - 2017.pdf: 16013047 bytes, checksum: 6d41859718e06adc6294f5de038bc631 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-03-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Considering the rate of increase in average annual temperature and the seasonality of rainfall in several regions of the country, investigations on the mechanisms of plant’s response to low water availability become relevant. Tabebuia Alliance species - monophyletic clade of the Bignoniaceae family are commonly known as ipe – are distribuited in areas with different soil and climatic conditions. This feature makes them an interesting model to understand mechanisms tolerance’s to abiotic stresses. For each species there were two groups: control group, which had maintained irrigation; low water availability group, which irrigation was stopped and the experiment continued until the subtrate reached 40% of field capacity. The main objective of this work was to identify differentially expressed genes (DEG) in four species - two from the Brazilian savannah (T. aurea and Handroanthus ochraceus) and two from seasonally dry forests (H. impetiginosus and H. serratifolius). Then, RNA was extracted from the plants for sequencing on the Illumina platform with paired-end sequences of 100 base pairs (bp). Sequences were evaluated for quality control and mapped onto the genome of H. impetiginosus to identify DEG using the R software. The DGEs obtained by DESeq2 were subjected to functional enrichment analysis and potential changes in the level of expression of genes encoding enzymes of particular metabolic pathways. In all tools, H. serratifolius species showed the highest number of DGE (4908 noDESeq2), while H. ochraceus had the lowest number of DGE (6 in DESeq2). Functional enrichment analyzes demonstrated that the species presented, individually or collectively, typical responses of low water availability such as decrease of photosynthetic rate, increase of proline and increase of starch degradation. Although species share some responses, the complexity of organisms does not allow them to exhibit identical behaviors. / Considerando-se o aumento crescente da temperatura média anual e a sazonalidade das chuvas em diversas regiões do país, investigações acerca dos mecanismos de resposta vegetais à baixa disponibilidade hídrica tornam-se pertinentes. Espécies da Aliança Tabebuia – clado monofilético da família Bignoniaceae são comumente conhecidas como ipê – possuem uma ampla distribuição, ocupando áreas com diferentes condições edafoclimáticas. Esta característica torna-as um modelo interessante para se entender os mecanismos de tolerância a estresses abióticos. Sob este cenário, o presente trabalho teve como principal objetivo a identificação de genes diferencialmente expressos (GDE) em quatro espécies – das quais duas são de cerrado stricto sensu (T. aurea e Handroanthus ochraceus) e duas de floresta estacional (H. impetiginosus e H. serratifolius). Para cada espécie houve dois grupos experimentais: grupo controle, o qual teve a irrigação mantida; grupo de baixa disponibilidade hídrica, o qual a irrigação foi interrompida e o experimento mantido até que o subtrato atingisse 40% da capacidade de campo. Em seguida, extraiu-se o RNA das plantas para sequencimento em plataforma Illumina com sequências do tipo “paired-end” de 100 pares de base (pb). As sequências foram avaliadas para o controle de qualidade e alinhadas no genoma de H. impetiginosus para identificação de genes diferencialmente expressos (GDEs) com o uso de pacotes do R. Os GDEs obtidos pelo DESeq2 foram submetidos a análise de enriquecimento funcional e potenciais alterações no nível de expressão de genes que codificam enzimas de determinadas vias metabólicas. Em todas as ferramentas, a espécie H. serratifolius apresentou o maior número de GDEs (4908 noDESeq2), enquanto H. ochraceus apresentou o menor número de GDEs (6 no DESeq2). As análises de enriquecimento funcional demonstraram que as espécies apresentaram, individual ou coletivamente, respostas típicas de baixa disponibilidade hídrica como diminuição de taxa fotossintética, aumento de prolina e aumento da degradação de amido. Ainda que as espécies compartilhem algumas respostas, a complexidade dos organismos não permite que exibam comportamentos idênticos.